Cellulose is a high molecular polysaccharide in which glucose molecules have been linked by β-1,4-glucoside linkage. Thus, hydrolysis of cellulose can produce glucose and so the cellulose can be effectively used as a glucose source. A substance that efficiently degrades cellulose and plays a key role in a series of reactions to extract the energy therefrom is cellulase.
Many cellulases have been isolated from fungi or bacteria. Cellulose is a less degradable substance, and degradation of cellulosic biomass by cellulase and utilization thereof are highly problematic, in terms of realization of practical applications thereof. Accordingly, elucidation of properties of such enzyme and efficient production of such enzyme are important themes associated with effective utilization of cellulase resources.
The intestinal symbiotic protists of termites were known to degrade cellulose with very high efficiency. However, such symbiotic protists were hard to culture, and there has not been progress with the analysis thereof for this reason. Even in recent years, only a small amount of research has been conducted on symbiotic protists and cellulases thereof.
In Japanese Patent Publication (kokai) No. H11-46764 A (1999) and in Watanabe, H., Noda, H., Tokuda, G., and Lo. N., 1998, Nature 394: 330-331, for example, cellulases produced by two types of termites (i.e., Reticulitermes speratus and Nasutitermes takasagoensis) are disclosed, although they are not derived from the symbiotic protists of termites. Such cellulases have a molecular weight of 40,000 to 50,000, thermostability of 60° C. or lower, an optimal pH of 5.0 to 6.0, and a specific activity on carboxy cellulose of 70 to 1,300 units/mg. The term “1 unit” used herein refers to the amount of an enzyme that generates a reducing sugar equivalent to 1 μmol of glucose per minute. The same definition applies hereinafter.
Japanese Patent Publication (kokai) No. 2003-70475 A discloses a protein having activity of cellulase derived from the symbiotic protists of coptotermes (Spirotrichonympha leidyi), which has a molecular weight of approximately 36 kDa, optimal pH 6.0, Vmax 148.2 units/mg, and Km 1.9 mg/ml.
Furthermore, the present inventors have made a report regarding evolution of the lignocellulose degrading system via a comprehensive technique based on detection of symbiotic bacteria, the transcription regulatory mechanism, molecular evolution of symbiotic organisms (i.e., Oxymondas), and EST analysis of the protists of termites (the Journal of the Japanese Society for Extremophiles, Vol. 4, No. 2, O-13, P-12 to P-15, 2005 (Jp)).